This invention relates generally to plant starch compositions, and concerns novel nucleotide sequences; polypeptides encoded thereby; vectors and host cells and host organisms comprising one or more of the novel sequences; a method of altering one or more characteristics of a plant; a plant having altered characteristics; starch obtained from such plants; and uses of the starch.
The majority of developments in cereal science in the recent past have concentrated primarily on the functionality of the gluten protein sub-units and their role in bakery systems. This has been greatly facilitated by the abundance of natural variation between cultivators for the gluten protein sub-unit components.
In contrast, although flour from commercially grown wheat varieties contains approximately 75-85% starch, the role of starch from a breeding perspective has been overlooked; this is largely due to the difficulty of measuring differences in starch structure. Of the limited amount of work that has been carried out however, there appears to be a lack of natural variation between different wheat cultivars. With the advent of recombinant DNA and gene transfer technologies it is now possible to create new variation in planta, therefore directly modifying starch composition in wheat becomes a realistic target.
Starch is the major form of carbon reserve in plants, constituting 50% or more of the dry weight of many storage organs, e.g. tubers, seeds of cereals. Starch is used in numerous food and industrial applications. In many cases, however, it is necessary to modify the native starches, via chemical or physical means, in order to produce distinct properties to suit particular applications. It would be highly desirable to be able to produce starches with the required properties directly in the plant, thereby removing the need for additional modification. To achieve this via genetic engineering requires knowledge of the metabolic pathway of starch biosynthesis. This includes characterisation of genes and encoded gene products which catalyse the synthesis of starch. Knowledge about the regulation of starch biosynthesis raises the possibility of xe2x80x9cre-programmingxe2x80x9d biosynthetic pathways to create starches with novel properties that could have new commercial applications.
The most significant property of starch derives from the ability of the native granular form to lose its order and to swell and absorb water upon suitable treatment, thereby conferring viscosity and texture, in a process known as gelatinisation. Gelatinisation has been defined (W A Atwell et al, 1988) as xe2x80x9c. . . the collapse (disruption) of molecular orders within the starch granule manifested in irreversible changes in properties such as granular swelling, native crystallite melting, loss of birefringence, and starch solubilisation. The point of initial gelatinisation and the range over which it occurs is governed by starch concentration, method of observation, granule type, and heterogeneities within the granule population under observationxe2x80x9d.
14 molecules of water per molecule of anhydrous glucose, i.e. a minimum of 75 % water, are necessary for full starch gelatinisation (Donovan, 1979). Starch gelatinisation is usually caused by heat, but can be caused by physical damage and some chaotropic agents, mainly dimethylsulphoxide (DMSO), urea, calcium chloride, strong base and acid.
The various events taking place during gelatinisation can be followed by various methods, including birefringence, X-ray diffraction, differential scanning calorimetry (DSC), 13C NMR. Swelling can be monitored by various methods, particularly rheology.
Differential scanning calorimetry (DSC) is a destructive method which records an endothermic event on heating of granules, generally thought to measure the temperature and the endothermic energy (delta H) required for the melting of the native crystallites. Starch gelatinisation temperature is independent of water content above 75% water (described as excess water), but increases when water is limited (Donovan, 1979).
The rate and extent of starch granule swelling upon heating dictate the type of viscosity development of aqueous starch suspensions on heating. Swelling behaviour is therefore of utmost technological importance. Viscosity increase on heating can be conveniently measured by a Brabender amylograph (Brabender is a Trade Mark) (Kennedy and Cabalda, 1991) or using a Rapid Visco analyser (Rapid Visco is a Trade Mark from Newport Scientific, Australia). FIG. 1 is a typical viscoamylgraph profile for wheat starch, produced in this way, showing changes in starch during and after cooking. As starch granules swell on uptake of water, in a process known as pasting, their phase volume increases, causing an increase in viscosity. The onset of pasting is indicated at A in FIG. 1. Peak viscosity, indicated at B in FIG. 1, is achieved when maximum phase volume is reached. Shear will then disrupt/cause fragmentation of the swollen granules, causing the viscosity to decrease. Complete dispersion is indicated at C in FIG. 1. This has been confirmed by an oscillatory rheology study of starch pastes at various stages of the viscosity profile (Svegmark and Hermansson, 1990). The viscosity onset temperature and peak viscosity are indicative of the initiation and extent of swelling, respectively. On cooling, leached amylose forms a network in a process involving reassociation of molecules, or retrogradation, causing an increase in viscosity as indicated at D in FIG. 1. Retrogradation (or set-back) viscosity is therefore indicative of the amount of amylose leached out of the granules.
The properties of wheat starch are useful in a large number of applications and also non-food (paper, textiles, adhesives etc.) applications. However, for many applications, properties are not optimum and various chemical and physical modifications well known in the art are undertaken in order to improve useful properties. Two types of property manipulation which would be of use are: the controlled alteration of gelatinisation and pasting temperatures; and starches which suffer less granular fragmentation during pasting than conventional starches.
Currently the only ways of manipulating the gelatinisation and pasting temperatures of starch are by the inclusion of additives such as sugars, polyhydroxy compounds of salts or by extensive physical or chemical pre-treatments. The reduction of granule fragmentation during pasting can be achieved either by extensive physical pre-treatments or by chemical cross-linking. Such processes are inconvenient and inefficient. It is therefore desirable to obtain plants which produce starch which intrinsically possesses such advantageous properties.
Starch consists of two main glucose polysaccharides: amylose and amylopectin. Amylose is a generally linear polymer comprising xcex1-1,4 linked glucose units, while amylopectin is a highly branched polymer consisting of an xcex1-1,4 linked glucan backbone with xcex1-1,6 linked glucan branches. In wheat endosperm amylopectin constitutes approximately 70% of the total starch content, with the balance being amylose. Amylopectin is synthesised through the concerted action of several enzymes, including soluble starch synthase(s) (SSS), starch branching enzyme(s) (SBE), starch de-branching enzyme(s) (DBE). The physical properties of starch are strongly affected by the relative abundance of amylose and amylopectin, therefore SSSs, SBEs and DBEs play a key role in determining both starch quantity and quality. As such, one approach to manipulating starch structure would be to modify the expression of the enzymes involved in starch biosynthesis in the endosperm using a transgenic approach.
SBE catalyses the formation of the xcex1-1,6 linkages, creating branch points in the growing starch molecule, via hydrolysis of an xcex1-1,4 linkage followed by reattachment of the released xcex1-1,4-glucan chain to the same or another glucosyl chain. This reaction also provides a new non-reducing end for further elongation of the original xcex1-1,4-glucan chain.
Multiple isoforms of starch branching enzyme have been described, biochemically, from a number of species including maize (Boyer and Preiss, 1978), rice (Nakamura et al., 1992), pea (Smith, 1988), potato (Khoshnoodi et al., 1993) and wheat (Morell et al., 1997). More recently, genomic and cDNA sequences for SBE have been characterised from several species including maize (Baba et al., 1991; Fisher et al., 1993; Gao et al. 1997) pea (Burton et al., 1995), potato (Kossmann et al., 1991), rice (Nakamura and Yamanouchi, 1992; Mizuno et al., 1993), Arabidopsis (Fisher et al., 1996), cassava (Salehuzzaman et al., 1992), and wheat (Rapellin et al., 1997, Nair et al., 1997, Rahman et al., 1997). Sequence alignment of these SBEs revealed a high degree of sequence conservation at the amino acid level and that the SBEs may be grouped into two distinct families, generally known as SBEI and SBEII. Further, analysis indicates that within a species there is generally of the order of 50% homology between the two families, SBEI and SBEII, while there is often greater homology within the two families between species.
Maize is unusual in that the maize SBEII family is thought to comprise two different members, known as SBEIIa and SBEIIb. There has been controversy over whether the SBEIIa and IIb enzymes are in fact a) encoded by genes at two different loci, and b) whether the genes represent different alleles at a single locus. Fisher et al (1996) and Gao et al (1997) have provided evidence that SBEIIa and SBEIIb are encoded by independent genes. However, there is no conclusive evidence that both isoforms exist together in any one maize genotype. The DNA clones for the two published gene sequences were purified from different genotypes of maize and it is thus possible that they represent different alleles of a single locus. In summary, in maize, three distinct SBE genes have been characterised to date (Baba et al., 1991; Fisher et al., 1993; Gao et al., 1997). SBEI is distinct from SBEIIa and SBEIIb in amino acid composition, substrate specificity, kinetic properties, and immunological reactivities, whereas SBEIIa and SBEIIb are similar in these respects (Guan and Preiss, 1993; Preiss 1991; Takeda et al., 1993). At the amino acid level the sequence exhibits approximately 50% homology with the SBEIIa and SBEIIb sequences, whereas SBEIIa and SBEIIb exhibit approximately 80% homology to each other.
Prior to the present invention, maize was unique in having SBEIIa- and SBEIIb-type enzymes. Although Arabidopsis has two SBEII family members, the sub-division in Arabidopsis does not appear to conform to that seen in maize: the Arabidopsis sub-family members do not obviously fall into the IIa and IIb categories as do the maize sequences. Both of the Arabidopsis SBEII genes have similar levels of homology to both the maize SBEII genes, SBEIIa and SBEIIb, but the similarities are not sufficient to be able to place the Arabidopsis genes into the same SBEIIa and SBEIIb categories as for maize. Indeed, the data, if anything, suggests that the Arabidopsis SBEII genes do not fall into the maize IIa and IIb categories. For barley, two forms of SBEII had been partly characterised. Although these have been called SBEIIa and SBEIIb, only a very limited amount of sequence information had been published (Sun et al, 1995) and it was not possible to infer or conclude that these forms correspond to the IIa and IIb categories of maize. In fact, based on the available barley sequence information both of the barley SBEII sequences (SBEIIa and SBEIIb) would appear to show greater homology to maize SBEIIa than to maize SBEIIb.
For all other plant species for which SBEII sequences have been identified and published, including potato, pea, rice, cassava, wheat and barley, no subdivision of the SBEII family comparable to the SBEIIa and SBEIIb division of maize has been made.
Studies of purified SBEI and SBEII demonstrate that these isoforms differ in their specificity for a substrate with respect to both chain length and degree of branching. In maize, SBEI and SBEII show distinct branching activities in vitro, with SBEI showing a higher rate of branching of an amylose substrate when compared to SBEII whereas both SBEIIa and IIb show higher rates of branching than SBEI when acting upon an amylopectin substrate (Guan and Preiss, 1993). Furthermore, maize SBEI preferentially transfers longer glucan chains (average chain length=24) than SBEII (average chain length=21(IIa) and 22(IIb)) (Takeda et al., 1993). A similar observation has been reported for SBEI and SBEII isoforms from wheat and pea (Morell et al., 1997; Smith, 1988). Mutational studies in maize, rice and pea demonstrate that high amylose mutants in each case are deficient in the branching enzyme activity analogous to maize SBEII (Martin and Smith, 1995; Morell et al., 1995). However, the linkage between the biochemical observations and the genetic evidence suggesting the differences in the roles remains unclear.
The present invention is based on the unexpected discovery of a novel class of SBEII genes in wheat, referred to herein as SBEII-1. The novel SBEII-1 gene sequence has strong homology with the maize SBEIIb gene. The wheat SBEII-1 genes are thought to be functionally equivalent to the maize SBEIIb gene, and on this basis it is believed that manipulation of the wheat SBEII-1 gene is likely to influence starch properties including starch gelatinisation temperature, in a manner analogous to manipulation of the maize SBEIIb gene as described in WO 97/22703.
In summary, although two different SBEII gene sequences are known from maize, Arabidopsis and barley, as discussed above, prior to the present invention there was no reason to expect that wheat would show a similar sub-division of SBEII genes as is seen for maize. The two Arabidopsis SBEII genes show a different sub-division, and prior to the present invention there was insufficient evidence to determine whether the two barley SBEII sequences belonged to the maize-type sub-division. That is, prior to the present invention there was no reason to expect that wheat would have two similar SBEII members comparable to those of maize. Subsequent to the present invention Sun et al (1998) have presented data which indicates that the barley sequences do indeed sub-divide in a similar manner to the maize SBEIIa and IIb sequences and the wheat SBEII-2 and SBEII-1 sequences discussed in this document.
The present inventors have used the high degree of sequence conservation between several SBE gene sequences to design oligonucleotide primers to motifs which are specific to either SBEI or SBEII families and have used these primers to amplify cDNA sequences from developing endosperm of wheat.
When this work was started, a single partial length wheat SBE cDNA clone had been reported (Mousley, 1994). Multiple sequence alignment of this wheat SBE sequence with other published SBE sequences from a number of plant species revealed a number of motifs which were highly conserved. Oligonucleotide primers designed to be complementary to these motifs were used to clone 3xe2x80x2 partial length cDNA clones of wheat SBEII. Alignment of the cDNA clone sequences indicated that the clones could be divided into two classes, which the inventors have designated SBEII-1 and SBEII-2, which showed greater than 90% similarity to members within a class but only 60% similarity between classes. Significantly, comparison between representative sequences from each class with previously identified wheat SBEII clones, pWBE6 (Mousley, 1994) and SBEII (Nair et al., 1997), showed that each appear to be homologues of the SBEII-2 class. The cloning of a wheat SBEII-1 cDNA is novel.
In one aspect the invention provides a nucleotide sequence encoding substantially the amino acid sequence shown in FIG. 10 (SEQ ID No: 2) or a functional equivalent of said nucleotide sequence.
The term functional equivalent is used in this context to encompass those sequences which differ in their nucleotide composition to that shown in FIG. 10 (SEQ ID No: 1) but which, by virtue of the degeneracy of the genetic code, encode polypeptides having identical or substantially identical amino acid sequences. It is intended that the term should generally apply to sequences which are sufficiently homologous to the sequence of the invention that they can hybridise to the complement thereof under stringent hybridisation conditions (eg as described by Sambrook et al 1989, ie washing with 0.1xc3x97SSC, 0.5% SDS at 68xc2x0 C.); such equivalents will preferably possess at least 86%, more preferably at least 90%, and most preferably at least 95%, sequence homology (ie sequence similarity) with the sequence of the invention. Sequence homology is suitably determined using the xe2x80x98MEGALIGNxe2x80x99 program of the software package DNAStar (MEGALIGN and DNAStar are Trade Marks). It will be apparent to those skilled in the art that the nucleotide sequence of the invention may also find useful application when present as an xe2x80x9cantisensexe2x80x9d sequence. Accordingly, functionally equivalent sequences will also include those sequences which can hybridise, under stringent hybridisation conditions, to the sequence of the invention (rather than the complement thereof). Such xe2x80x9cantisensexe2x80x9d equivalents will preferably possess at least 86%, more preferably at least 90%, and most preferably 95% sequence homology with the complement of the sequence of the invention.
In another aspect, the invention provides a nucleotide sequence comprising substantially the sequence of B2 shown in FIG. 3 (SEQ ID No: 3), or a functional equivalent thereof.
In a further aspect, the invention provides a nucleotide sequence comprising substantially the sequence of B4 shown in FIG. 3 (SEQ ID No: 4), or a functional equivalent thereof.
Another aspect of the invention provides a nucleotide sequence comprising substantially the sequence of B10 shown in FIG. 3 (SEQ ID No: 5), or a functional equivalent thereof.
Yet a further aspect of the invention provides a nucleotide sequence comprising substantially the sequence of B1 shown in FIG. 3 (SEQ ID No: 6), or a functional equivalent thereof.
In another aspect the invention provides a nucleotide sequence encoding substantially the amino acid sequence of B6 shown in FIG. 4 (SEQ ID No: 7), or a functional equivalent thereof.
The term functional equivalent in this context has the same general meaning as discussed above, although equivalents for B2, B4, B10 and B6 will preferably possess at least 90%, more preferably at least 95%, sequence homology with the relevant sequence of the invention, while equivalents for B1 will preferably possess at least 97% sequence homology with the sequence of the invention.
The sequences of the invention are part of novel wheat SBEII genes, with B1 being a novel subclass of the known class of SBEII genes, referred to herein as SBEII-2, with the novel subclass being called SBEII-2B. The remaining sequences are all of a completely new class of wheat SBEII genes, referred to herein as SBEII-1. The sequences have been found to fall into 3 sub-classes, to be discussed below.
The novel wheat SBEII-1 genes that are the subject of this invention have strong sequence homology with the maize SBEIIb gene. The wheat SBEII-1 genes are thought to have similar functional properties to the maize SBEIIb gene. On this basis it is expected that by genetic manipulation of the wheat SBEII-1 gene it will be possible to influence properties of starch produced by a plant, including the gelatinisation temperature and rheological properties of starch, in a manner analogous to manipulation of the maize SBEIIb gene described in WO 97/22703. The content of WO 97/22703 is incorporated herein by reference.
The present invention also includes within its scope a portion of any of the above sequences, comprising at least 500 base pairs and having at least 90% sequence homology to the corresponding portion of the sequence from which it is derived.
Although the coding sequences of the novel wheat SBEII-1 genes have strong sequence homology with the maize SBEIIb gene, there is much greater divergence in the 3xe2x80x2 untranslated parts of the sequences, with a maximum of 31.8% homology between the 3xe2x80x2 untranslated sequences of wheat SBEII-1 and maize SBEIIb as is apparent from FIG. 8.
In another aspect the invention thus provides a nucleotide sequence comprising substantially the sequence shown in FIG. 5 (SEQ ID No: 8), FIG. 6 (SEQ ID No: 9) or FIG. 7 (SEQ ID No: 10), or a functional equivalent thereof.
The term functional equivalent in this context has the same general meaning as discussed above, but with equivalents preferably at least 32%, more preferably at least 40%, 50%, 60%, 70%, 80% or 90% sequence homology with the sequence of the relevant Figure.
It is thought such 3xe2x80x2 untranslated sequences may be useful, both in sense and antisense function, in manipulation of starch properties by affecting SBE expression in plants, as will be discussed below.
The sequence may include further nucleotides at the 5xe2x80x2 or 3xe2x80x2 end. For example, for ease of expression, the sequence desirably also comprises an in-frarne ATG start code, and may also encode a leader sequence.
The invention also covers a nucleic acid construct comprising a nucleotide sequence or portion thereof in accordance with the invention conveniently operably linked, in sense or antisense orientation, to a promoter sequence.
Also included within the scope of the invention is amino acid sequence encoded by any of the nucleotide sequences of the invention.
The invention also provides vectors, particularly expression vectors, comprising the nucleotide sequence of the invention. The vector will typically comprise a promoter and one or more regulatory signals of the type well known to those skilled in the art. The invention also includes provision of cells transformed (which term encompasses transduction and transfection) with a vector comprising the nucleotide sequence of the invention.
Nucleotide sequences in accordance with the invention may be introduced into plants, particularly but not exclusively wheat plants, and it is expected that this can be used to affect expression of SBE in the plant and hence affect the properties of starch produced by the plant. In particular, use of sequences in antisense orientation is expected to reduce or suppress enzyme expression. Additionally, it has recently been demonstrated in other experimental systems that xe2x80x9csense suppressionxe2x80x9d can also occur (i.e. expression of an introduced sequence operably linked in the sense orientation can interfere, by some unknown mechanism, with the expression of the native gene), as described by Matzke and Matzke 1995. Any one of the methods mentioned by Matzke and Matzke could, in theory, be used to affect the expression in a host of a homologous SBE gene.
It is believed that antisense methods are mainly operable by the production of antisense mRNA which hybridises to the sense mRNA, preventing its translation into functional polypeptide, possibly by causing the hybrid RNA to be degraded (e.g. Sheehy et al., 1988; Van der Krol et al.,). Sense suppression also requires homology between the introduced sequence and the target gene, but the exact mechanism is unclear. It is apparent however that, in relation to both antisense and sense suppression, neither a full length nucleotide sequence, nor a xe2x80x9cnativexe2x80x9d sequence is essential. Preferably the xe2x80x9ceffective portionxe2x80x9d used in the method will comprise at least one third of the full length sequence, but by simply trial and error other fragments (smaller or larger) may be found which are functional in altering the characteristics of the plant.
Thus, in a further aspect the invention provides a method of altering the characteristics of a plant, comprising introducing into the plant an effective portion of the sequence of the invention operably linked to a suitable promoter active in the plant so as to affect expression of a gene present in the plant. Conveniently the sequence will be linked in the antisense orientation to the promoter. Preferably the plant is a wheat plant. Conveniently, the characteristic altered relates to the starch content and/or starch composition of the plant (i.e. amount and/or type of starch present in the plant). Preferably the method of altering the characteristics of the plant will also comprise the introduction of one or more further sequences, in addition to an effective portion of the sequence of the invention. The introduced sequence of the invention and the one or more further sequences (which may be sense or antisense sequences) may be operably linked to a single promoter (which would ensure both sequences were transcribed at essentially the same time), or may be operably linked to separate promoters (which may be necessary for optimal expression). Where separate promoters are employed they may be identical to each other or different. Suitable promoters are well known to those skilled in the an and include both constitutive and inducible types. Examples include the CaMV 35S promoter (e.g. single or tandem repeat) and the ubiquitin promoter. Advantageously the promoter will be tissue-specific. Desirably the promoter will cause expression of the operably linked sequence at substantial levels only in the tissue of the plant where starch synthesis and/or starch storage mainly occurs.
The sequence of the invention, and the one or more further sequences if desired, can be introduced into the plant by any one of a number of well-known techniques (e.g. Agrobacterium-mediated transformation, or by xe2x80x9cbiolisticxe2x80x9d methods). The sequences are likely to be most effective in affecting SBE activity in wheat plants, but theoretically could be introduced into any plant. Desirable examples include pea, tomato, maize, rice, barley, sweet potato and cassava plants. Preferably the plant will comprise a natural gene encoding an SBE molecule which exhibits reasonable homology with the introduced nucleic acid sequence of the invention.
In another aspect, the invention provides a plant cell, or a plant or the progeny thereof, which has been altered by the method defined above. The progeny of the altered plant may be obtained, for example, by vegetative propagation, or by crossing the altered plant and reserving the seed so obtained. The invention also covers parts of the altered plant, such as storage organs. Conveniently, for example, the invention covers grain comprising altered starch, said grain being obtained from an altered plant or the progeny thereof. Grain obtained from altered plants (or the progeny thereof) will be particularly useful materials in certain industrial applications and for the preparation and/or processing of foodstuffs and may be used, for example, in bakery products.
In particular relation to wheat plants, the invention provides a wheat plant or part thereof which, in its wild type possesses an effective SBEII-1 gene, but which plant has been altered such that there is either reduced, increased or no effective expression of an SBEII-1 polypeptide within the cells of at least part of the plant. The plant may have been altered by the method defined above, or may have been selected by conventional breeding to be deleted for the SBEII-1 gene, the presence or absence of which can be readily determined by screening samples of the plants with a nucleic acid probe or antibody specific for the wheat gene or gene product respectively.
The invention also provides starch extracted from a plant altered by the method defined above, or from the progeny of such a plant, the starch having altered properties compared to starch extracted from equivalent, but unaltered, plants. The invention further provides a method of making altered starch, comprising altering a plant by the method defined above and extracting therefrom starch having altered properties compared to starch extracted from equivalent, but unaltered, plants. It is believed that use of nucleotide sequences in accordance with the invention will enable the production of starches, particularly wheat starches, having a wide variety of novel properties. For example, it may be anticipated that plants altered to give a reduction in SBEII activity will give rise to a starch with a relatively higher proportion of amylose and a lower proportion of amylopectin compared with that from unaltered plants.
In particular the invention provides the following: a plant (especially a wheat plant) altered by the method defined above, containing starch which, when extracted from the plant, has an elevated gelatinisation onset and/or peak temperature as measured by DSC, compared to starch extracted from a similar, but unaltered, plant; a plant (especially a wheat plant) altered by the method defined above, containing starch which, when extracted from the plant, has a elevated gelatinisation onset temperature (conveniently elevated by at least 3xc2x0 C., possibly by at least 7xc2x0 C., by at least 12xc2x0 C. or possibly even by 15 to 25xc2x0 C.) as measured by DSC compared to starch extracted from a similar, but unaltered plant; a plant (especially a wheat plant) altered by the method defined above, particularly to reduce expression of SBEII-1 polypeptide, containing starch which, when extracted from a plant, has a higher amylose:amylopectin ratio compared to starch extracted from a similar, but unaltered plant.
The present invention particularly covers starch extracted from a plant altered by the method of the invention, particularly starch having an increased gelatinisation temperature. Such starch is useful, eg in bakery products, having particular benefits in certain situations, and the invention also covers products, particularly bakery products, made from such starch. The invention also covers starch extracted from a plant altered by the method of the invention and having an increased amylose:amylopectin ratio.
The invention will be further described, by way of illustration, in the following Examples and with reference to the accompanying drawings, in which: